Membrane cofactor protein (MCP, CD46) is a complement regulatory protein that binds C3b andC4b and serves as a cofactor for their limited protedytic degradation. A major developmentduring the past grant cycle was the discovery that mutations in CD46 predispose to atypical hemotytlc uremic syndrome (aHUS). This finding has an Immediate impact on treatment options since renal transplantation would be curative in CD46 deficiency. This is especially significant since aHUS can be a life-threatening condition that recurs in patients (usually youngchildren) with about 50%developing renal failure. Atypical HUSis now recognized as a disease of complement deregulation due to mutations in complement regulatory proteins. We will address how CD46 deficiency prodtepoeesto aHUS. A major goal of this grant is to characterize CD46's regulatory activity in situ. In patients with aHUS and their families, we will 1) establish a facility to identify mutations and determine the functional repertoire of the mutant proteins;2) use model systems(CHOcells expressing the mutant proteins, EB virus transformed human B lymphocytes from aHUS families, and human endothelial cells), assess Inhibitory activity in situ;RNAiwill be employed to create cells with a specific inhibitor deficiency state;3) employ scFv-complement regulator(s) chimeras to target inhibitors to RBCs and endothelial cells in order to correct the deficiency and to define the most potent inhibitory mix of regulators to block complement activation;4) monitor membrane movements of regulators as they are cross-linked with Abs and pathogens and in response to complement activation. A theme underlying these specific alms is to explorethe process whereby the host limits complement activation on altered and injured self cells. This phenomenon wehave termed TRACS, for targeted and restricted activation of the complement system, is little studied. We propose that the profile of complement activation on self-tissue has unique purposes and distinct features compared to its activation on microbes. Our long term goal is to understand how this is accomplished using CD46 deficiency In aHUS as the Illustrative example. Our proposal will help define how deficiency of the complement regulator CD46 predisposes to human disease hemolytic uremic syndromeas well as develop ways to deliver regulators to treat such conditions. PERFORMANCE SlTE(6) (orrjinizjitlon, city, [unreadable]al*) Washington University, St. Louis, Missouri PHS 398(Rev.Oft/04) P*0*2 FormP[unreadable]o[unreadable]J Principal Investigator/Program Director (Last, First, Middle): Atkinson, John P. KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Atkinson, John P. Washington University Principal Investigator Bertram, Paul Washington University Sr. Research Scientist Gaudy, Amy Washington University Post-Doc Trainee Hourcade, Dennis E. Washington University Res. Assoc. Professor Leung, Marilyn Washington University Staff Scientist Liszewski, M. Kathryn Washington University Co-Investigator Spitzer, Dirk Washington University Research Associate OTHER SIGNIFICANT CONTRIBUTORS Name Bessler, Monica Fremeaux-Bacchi, Veronique Goodship, Timothy H.J. Halperin, Jose "Kavanagh, David *Richards, Anna Wilson, Richard K. "joining Atkinson lab 4/1/05 Organization Washington University Hosp. Europeen Georges Pompido University of Newcastle Upon Tyne Brigham &Women's Hospital Washington University Washington University Washington University Role on Project Collaborator Collaborator Collaborator Collaborator Research Associate Research Associate Collaborator Human Embryonic Stem Cells LjU No I I Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.gov/registry/index.asp. Use continuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. DYes No PHS 398 (Rev. 09/04) Page 2 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixessuch as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Atkinson, John P. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,